1. Field of the Invention
The present invention relates generally to the fields of protein chemistry, immunology, therapeutic pharmaceuticals, and vaccine development. More specifically, the present invention discloses crystal structure of Staphylococcus aureus clumping factor A (ClfA) in complex with fibrinogen (Fg) derived peptide and its use in the design of ClfA targeted vaccines and therapeutic agents (including monoclonal antibodies).
2. Description of the Related Art
Staphylococcus aureus is a Gram-positive commensal organism that permanently colonizes 20% of healthy adults and transiently colonizes up to 50% of the population (1). For many years, S. aureus has been a major nosocomial pathogen causing a range of diseases from superficial skin infections to life-threatening conditions, including septicemia, endocarditis and pneumonia (1-2). Within the last decade an increasing number of invasive infections caused by community-acquired S. aureus have been recorded in otherwise healthy children and young adults (3-4). The continued emergence of antibiotic resistance among clinical strains has made the treatment of staphylococcal infections challenging, underscoring the need for new prevention and treatment strategies (1).
A detailed characterization of the molecular pathogenesis of S. aureus infections may expose new targets for the development of novel vaccines and therapeutics. Several staphylococcal virulence factors have been identified including capsule, surface adhesins, proteases, and toxins (5-8). One of these virulence factors is the MSCRAMM clumping factor A (ClfA). ClfA is the major staphylococcal fibrinogen (Fg) binding protein and is responsible for S. aureus clumping in blood plasma (9-10). Essentially all S. aureus clinical strains carry the clfA gene (11); ClfA is a virulence factor in a mouse model of septic arthritis (12) and in rabbit and rat models of infective endocarditis (13-15).
ClfA generates strong immune responses and has shown potential as a vaccine component in active and passive immunization studies. In one study, mice vaccinated with a recombinant ClfA segment containing the Fg-binding domain and subsequently infected with S. aureus showed significantly lower levels of arthritis (12). In another study, mice passively immunized with polyclonal or monoclonal antibodies against the ClfA Fg-binding domain were protected in a model of septic death (16). The humanized monoclonal antibody, Aurexis®, has a high affinity for ClfA and inhibits ClfA binding to Fg (17). Aurexis is currently in clinical trials in combination with antibiotic therapy for the treatment of S. aureus bacteremia (18).
ClfA belongs to a class of cell wall-localized proteins that are covalently anchored to the peptidoglycan (6, 19-20). Starting from the N-terminus, ClfA contains a signal sequence followed by the ligand-binding A region composed of three domains (N1, N2, and N3), the serine-aspartate repeat domain (R region), and C-terminal features required for cell wall anchoring such as the LPXTG motif, a transmembrane segment and a short cytoplasmic domain (21-23). A crystal structure of a Fg-binding ClfA segment (residues 221-559) which includes two of the domains (N2N3) demonstrates that each domain adopts an IgG-like fold (24). This domain architecture was also determined from the crystal structure of the ligand binding segment of the Staphylococcus epidermidis SdrG, an MSCRAMM that binds to the N-terminal region of the Fg β-chain (25).
Molecular modeling and sequence analysis indicated that the staphylococcal Fg binding MSCRAMMs ClfB and FnbpA could also have a structural organization similar to that of SdrG and ClfA, setting the stage for a common mechanism of ligand binding. For SdrG, a dynamic mechanism of Fg binding termed “Dock, Lock and Latch” (DLL) has been proposed based on a comparison of the crystal structures of SdrG N2N3 as an apo-protein and in complex with a synthetic peptide mimicking the targeted site in Fg (25). In the SdrG DLL model, the apo-form of the protein adopts an open conformation that allows the Fg ligand access to a binding trench between the N2 and N3 domains. As the ligand peptide docks into the trench, a flexible C-terminal extension of the N3 domain is redirected to cover the ligand peptide and “lock” it in place. Subsequently the C-terminal part of this extension interacts with the N2 domain and forms a β-strand complementing a β-sheet in the N2 domain. This inserted β-strand serves as a latch to form a stable MSCRAMM ligand complex.
ClfA binds to the C-terminus of the Fg γ-chain (9, 23) and a synthetic 17 amino acid peptide corresponding to this region was shown to bind to ClfA. Interestingly, the A-region of another staphylocccal MSCRAMM FnbpA protein and human platelet αIIbβ3 integrin also binds to the same region in Fg (23, 26-28). A recombinant form of ClfA has been shown to inhibit platelet aggregation and the binding of platelets to immobilized Fg (9). Although the individual N2 and N3 sub-domains in SdrG and ClfA are structurally similar, the overall orientation of one with respect to the other is different.
Thus, prior art is deficient in structural characterization of how ClfA binds Fg and its use in the design of vaccines and therapeutic compounds for the prevention and treatment of staphylococcal infections. The current invention fulfils this long standing need in the art.